The long-term objective of my research centers on elucidation of the mechanisms whereby extra-cellular signals are sensed by the cell cycle machinery and regulate cell cycle progression. This information will provide the framework necessary to elucidate how growth regulatory pathways are subverted in neoplasia. Our current studies focus on how growth-signaling pathways regulate the mitogenically responsive D-type cyclins and more specifically how these pathways regulate accumulation of an active, nuclear cyclin D1- dependent kinase. The importance of elucidating the mechanisms that regulate nuclear accumulation of cyclin D1 is emphasized by our demonstration that the failure of the cell to remove active D1/CDK complexes from the nucleus during S-phase results in cell transformation. Cyclin D1 accumulates in the nucleus during G1 phase of the cell cycle in response to mitogenic stimulation. During S-phase, cyclin D1 is targeted to the cytoplasm via phosphorylation of the at a single threonyl residue, Thr-286, by GSK-3beta. We have identified a naturally occurring cyclin D1 isoform, D1b, which lacks critical residues necessary for cyclin nuclear export. Our preliminary data indicate that this isoform is specifically expressed in cancer cells and is likely to represent an oncogenic variant of the canonical cyclin D1 isoform. In addition, mutations in the C-terminus of cyclin D1 that will disrupt cyclin D1 nuclear export have been reported in endometrial cancer. We hypothesize that cyclin D1b and mutant nuclear cyclin D1 isoforms are oncogenic variants of canonical cyclin D1 (D1a) whose overexpression contributes directly to neoplastic malignancy. To test this hypothesis, we propose to: 1) Determine the frequency of cyclin D1b overexpression in breast carcinoma; 2) Determine the capacity of cyclin D1 mutants that are constitutively nuclear to drive mammary carcinoma; and 3) Characterize cancer specific cyclin D1 mutants. To accomplish these goals, we will utilize resources here at the University of Pennsylvania to determine the frequency of the nuclear cyclin D1 variant, D1b, in primary human breast cancer and assess its prognostic value. In addition, we will use newly established mouse models to assess the oncogenicity of nuclear cyclin D1 isoforms in vivo. Finally, we propose to characterize newly identified cyclin D1 mutants for their ability to undergo active, CRM1-dependent nuclear export. While expression of wild-type cyclin D1 is not overtly oncogenic in vitro or in animal models, my laboratory has demonstrated that constitutively nuclear cyclin D1 isoforms are strongly oncogenic. Cancer Relevance. Findings from this work would support a model wherein constitutively nuclear cyclin D1 functions as an initiating oncogene and that mechanisms, which regulate its nuclear retention will be targeted during carcinogenesis. Consistent with this, in our preliminary data we provide evidence for a novel, constitutively nuclear cyclin D1 isoform, which is expressed exclusively in cancer. The work proposed herein will establish the relationship between cyclin D1 nuclear retention and the function of cyclin D1 as an oncogenic protein in mammary carcinoma.